Quantitative single cell monitoring of protein synthesis at subcellular resolution using fluorescently labeled tRNA.

ABSTRACT: We have developed a novel technique of using fluorescent tRNA for translation monitoring (FtTM). FtTM enables the identification and monitoring of active protein synthesis sites within live cells at submicron resolution through quantitative microscopy of transfected bulk uncharged tRNA, fluorescently labeled in the D-loop (fl-tRNA). The localization of fl-tRNA to active translation sites was confirmed through its co-localization with cellular factors and its dynamic alterations upon inhibition of protein synthesis. Moreover, fluorescence resonance energy transfer (FRET) signals, generated when fl-tRNAs, separately labeled as a FRET pair occupy adjacent sites on the ribosome, quantitatively reflect levels of protein synthesis in defined cellular regions. In addition, FRET signals enable det

SUBMITTER: Barhoom S 

PROVIDER: S-EPMC3201886 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature